Conserved virulence factor A (CvfA) contains a metal-dependent phosphohydrolase domain and regulates the expression of glycolytic enzymes and virulence factors of Streptococcus pyogenes in response to low nutrient conditions. Group A Streptococcus (GAS) is associated with a broad range of human diseases including scarlet fever, impetigo, pharyngitis, necrotizing fasciitis, streptococcal toxic shock syndrome and the post- streptococcal sequelae of rheumatic fever. The growth phase dependent regulation of protein expression allows for adaptation to various host tissues and represents a potential switch in the mode of infection reflected in the change in virulence factor expression. Our overall goal is to understand the mechanism of gene regulation by defining the catalytic activity, the RNA specificity and the nutrient-dependent signaling response of CvfA. Our previous studies indicated that CvfA is associated with the glycolytic enzyme, enolase, which may be moonlighting as a nutrient-sensing regulator of CvfA activity. We also showed that in low-carbohydrate media, CvfA down-regulates the expression of the virulence factors M protein, streptokinase and CAMP factor, while up-regulating expression of the secreted protease, SpeB. Preliminary studies show that purified recombinant CvfA protein has endonuclease activity that specifically cleaves the mRNA transcript of M protein, while showing no effects on the mRNA transcript of RecA. Our working hypothesis is that CvfA is a sequence- specific endonuclease that utilizes an RNA-binding KH domain to target mRNA transcripts for cleavage by the histidine-aspartate containing (HD) phosphohydrolase domain resulting in regulation of gene expression through RNA degradation. Our specific aims are to: 1) determine the structure and RNA binding specificity of the KH domain, 2) identify the mRNA substrates of CvfA and define the nutrient dependent regulatory role of CvfA, and 3) determine the metal-dependent catalytic activity and structure of the HD domain. Our approach combines structural studies and functional assays including NMR spectroscopy to study the KH and HD domain structures, catalytic assays to define the mechanism of phosphohydrolase activity, mRNA decay assays to elucidate the nutrient signaling response, mRNA cleavage assays and in vitro selection to define the sequence specificity of CvfA. The impact of our results will lead to new therapeutic strategies against streptococcal infections following three potential approaches: 1) interference with RNA recognition by the KH domain, 2) down regulation of nutrient signaling pathways, and 3) developing inhibitors of phosphohydrolase activity based on lead compounds identified through catalytic assays. PUBLIC HEALTH RELEVANCE: Streptococcus pyogenes is an important human pathogen against which new therapeutic methods must be developed urgently since safe vaccines do not exist. CvfA influences the expression of genes controlling virulence and nutrient utilization in response to nutrient availability. We will study the structure and cellular function of CvfA in ordr to understand the mechanism of the virulence control. This will allow us to begin developing therapeutic measures against streptococcal diseases based on our knowledge of CvfA.